Lecture 21 - DNA Replication

Question 1

How can DNA replication begin ?

Answer 1

DNA is semi-conervative - in order for replication 2 strands must first be separated

Each strand used as a template to determine order of nucelotides that will be synthesised in complementary strands

Makes 2 double helix molecules, in each there is one old strnd and one new strand

Question 2

What direction does DNA synthesis occur in?

Answer 2

DNA synthesis ALWAYa occurs in a 5’ -> 3’ direction by the formation of phosphodiester bonds.

Template strand and prime strnd are anti-parralel

Question 3

How is a new strand of DNA produced during replication?

Answer 3

The new strand is produced by extension of the prime strnd. Done by polymerisation of new nucleotide ontoi 3’ hydroxyl end.]

Chemical rxn here is a nucleophilic attack on the phosphate of the new DNTP

This binds the nucleotide into place int primary strna and release pyrophaisphete

Question 4

What is meant by a nucleophilic attack ?

Answer 4

The transfer of electrons to form a diester bond.

Question 5

Why is the anti-parallel nature of DNA a problem for DNA replication?

Answer 5

DNA synthesis can only proceed in a 5 to 3 prime direction but on the other lagging strand, DNTP is being added to 5 prime. This is wrong.

Question 6

How does DNA replication solve the problem of DNTP trying to attach to the 5’ end of a lagging DNA strand?

Answer 6

Okozaki fragments allow DNA polymerase to bind to the prime side on the lagging strand

Question 7

What are E.coli and human replication forks?

Answer 7

Their key components are highly conserved

Question 8

Whta is thw first step of DNA replication

Answer 8

Formation of the replcation fork where the paired DNA strands are separated by DNA helicase

Question 9

What is the second step in DNA replication ?

Answer 9

Binding of primers to 3’ hydroxyl end.
]Acts as a template for DNA polymerase to bind to and DNA synthesis of new strand.

Question 10

What does the antiparalle orientation of parental strnas mean ?

Answer 10

Means a unidirectional orientation of new DNA synthesis (5’ - 3’) means that both new strands cannot be synthesised continuously

Question 11

What is the difference between the leading and lagging strand ?

Answer 11

Leading Strand - CONTINUOS 5 to 3 prime
Lagging strand - discontinuous also 5 to 3 prime direction

Question 12

What are Okazaki fragments ?

Answer 12

RNA primers which binf to DNA by DNA primase and act as a template for DNA polymerase

They are short fragments of DNA

Question 13

WHat happens in third step of DNA replication ?

Answer 13

Primers allow DNA polymerase to bind and start synthesis as a new strnad.

DNA primase makes these primers (RNA DNA hybrids complementary to template) in order to allow DNA polymerase to bind and extend by adding nucleotides onto the 3’ hydroxyl end. This happens on the leading strand, lagging strand is different.

Question 14

How do the following synthesise a continuous DNA strand from lagging strand and Okazaki fragments ?

Answer 14

DNA polymerase - extends RNA primer - requires a Primer-Template junction

Ribonuclease H - removes RNA primer

DNA Polymerase - extends across gap

DNA ligase seals the nick

Question 15

How does DNA Helicase use ATP ?

Answer 15

Uses ATP to separate parental DNA strands at the Replication Fork and move Replication Fork forward.

Breaks H bonds between strands

ATP -> ADP + Pi

Question 16

What happens when there is a mutation in genes encoding DNA helicase?

Answer 16

Causes Werner Syndrome - autosomal recessive

Premature aging disorder (progeria)

Caused by a deficiency in WRN gene, that encodes the DNA helicase RECQ.

Cells Senscencing, become permanently arrested.

Question 17

What is Bloom Syndrome?

Answer 17

Bloom Syndrome - a rare cancer syndrome caused by loss-of-function mutations in a RecQ-family DNA helicase which maintains genome integrity.

Bloom suppresses illigetamte homologous recombination and it is involved in removing roadblocks that can cause disruption to Replication Fork.

Extreme sensitivity to sun exposure and red on the face.

Question 18

What is the processivity of DNA Polymerase?

Answer 18

An enzymes ability to catalyse cosecutive rxns without releasing its substrate. These enzymes are processing.

The degree of processivity is the amount of nucleotides added each time the enzyme binds to a template.

Avg Polyemrase needs 1 sec to bind. Non - processing - adds one nucleotide per sec

Processive - adds multiple nucleotides per sec

Question 19

How does the sliding of DNA polyemerase along the template allow increased pROCESSIVITY?

Answer 19

Sliding Clamp (ATP-dependent) - positioned close to primer, binds to primer-tempopate junction with help from a clamp loader.

With the release of ATP clamp loader is released and the Polymerase is clamped onto template strand and it recruits the Polymerase and keeps it locked onto strand. Aloows it to increase and continue processivity

Question 20

How does protein SSB aide the processivity and function of DNA Polymerase?

Answer 20

1. As helicase unwinds the DNA it makes single sections od DNA in the leading and laging strand
2. If the DNA is near regions similar the DNA starts to bind back to each other. These are called hairpins which can block the Preocessivity.
3. The SSBs bind to single stranded DNA and keep it from forming hair pins.

Question 21

How does the enzyme DNA Topoisomerase prevent DNA being tangled and enhance processivity?

Answer 21

They prevent DNA getting tangled during replcation

The remaining double strand helix of original DNA during replicatio can get tangled. creating torsional strength.

Topoisomerases transiently nicking and resealing the backbone of the parental helix to release tension.

Question 22

What are the 2 types of DNA Topoisomerases?

Answer 22

Type 1 - nick and reseal one of the 2 DNA strands, no ATP required
Type 2 - Nick and reseal both strands. ATP required

Question 23

What is the orgins of replication ?

Answer 23

E.coli used because it os conserved in humans. Only 1 origin in E.coli but +100,000 in humans

Begins at oriC, specific DNA sequences that recruit replication initiator proteins that bind to origin and promote melting of double strand DNA.

Provides access for DNA replication machinery and creaes replication forks.

Question 24

How is integreity reserved in genome ?

Answer 24

Biphasic

Each origin of replication must fire only once per cell cycle

Question 25

What are the 2 biphasic steps in the initiation of DNA replication?

Answer 25

1. Replicator Selection occurs in G1 - forms a pre-Replicative Complex
2. Origin Activation occurs in S phase - unwinding of DNA and recruitment of DNA Polymerase

Question 26

What does the temporal seperation of Replicator selection and origin activation?

Answer 26

Ensures that each origin is used and each chromosome is only replicated exactly once per cell cycle.

Question 27

How does the eukaryotic Replicator Selection occur in G1 in yeast and lead to formation of a Pre-Replicative Complex (pre-RC)?

Answer 27

1. Origin Recognition Complex (ORC) binds to Replicator sequence, e.g ARS sequence in yeast
2. Helicased-loading proteins Cdc6 and Cdt1 bind to ORC
3. Recruits helicase Mcm2-7 binds to complete formation of pre-RC

INACTIVE - Doesnt start replicated only loaded onto DNA

Question 28

How is pre-RC regulated?

Answer 28

High lvls of Cdk activity in S phase activates existing pre-RC but orevents formation of new pre-RCs and replication can star

Cdk lvls and cell cycle ensures that chromosomes are replicated exactly once per cell cycle

Question 29

When are the Cdk levels high or low in the cell cycle?

Answer 29

Low Cdk level in G1 phase

High Cdk level in S, M and G2 phase

Question 30

How is replication finished ?

Answer 30

DNA Polymerase and Ligase close all but one gap.

RNA primer at end of chrmomsome, once it is removed by RNA H there will be a gap that cannot be synthesised.

Produces an SS overhang (3 prime) unreplicated DNA. One is on the other strand too

Question 31

Whta does RNA H do?

Answer 31

Removes RNA primers Sshorteing ends and making 3 prie overhangs.

The shortening could lead to loss of viable coding info -> premature aging and cancer

Question 32

How do chromosomes stop DNA from being lost?

Answer 32

Non-coding telemere regions occur at ends of chromosomes to stop =information from being lost.

They are created by telomerase. Telomerase will add the telomere repeat ‘TTAGGG’ onto ends of every replicated DNA molecule to extend the 3 prime end.

Eventualy will be long enough to allow DNA Primase to bind and initiate new RNA primer synthesis which can then be extended as an extra Ozaki fragment by DNA polymerase

Question 33

How does Telomerase know to add TTAGGG repeats?

Answer 33

Telomerase is a ribonucleoprotein, contains both RNA and protein subunits.

RNA component contains sequence AUCCCAAUC which can base pair with the ttaggg TEOLMERE repeat.

Telomerase can use reverse transcriptase activity to polyemrase DNA from its RNA template in a process called the Telomere shuffle.

Question 34

How does the Telomerase shuffle work ?

Answer 34

1. Telomerase RNA airs with existing telomere repeat with 3 orime hang at the end
2. Telomerase uses it as a template to reverse transcribe the 3 bases (adds TTA) shuffles along
3. 3 -> 6 ->6 synthesised this many each time and moves along.